Process for the production of l-aspartic acid

ABSTRACT

At least one monomer selected from the group consisting of acrylamide, N,N&#39;&#39;-lower alkylene-bis(acrylamide) and bis (acrylamidomethyl)ether is polymerized in an aqueous suspension containing an aspartase-producing microorganism. The resultant immobilized aspartase-producing microorganism is subjected to enzymatic reaction with ammonium fumarate or a mixture of fumaric acid or its salt and an inorganic ammonium salt. Divalent metal ion is preferably added to the anzymatic reaction solution. Laspartic acid is produced.

United States Patent [191 Chibata et a1.

[ Feb. 12, 1974 1 PROCESS FOR THE PRODUCTION OF L-ASPARTIC ACID [75]Inventors: lchiro Chibata, Osaka; Tetsuya Tosa, Kyoto; Tadashi Sato,Osaka,

all of Japan [73] Assignee: Tanabe Seiyaku Co., Ltd., Osaka,

Japan [22] Filed: Oct. 18, 1972 [2]] Appl. No.: 298,529

[30] Foreign Application Priority Data Oct, 28, 1971 Japan 46-85778 [52]US. Cl 1 95/30, 195/DIG. 11, 195/59 [51] Int. Cl Cl2d 1/02 [58] Field ofSearch 195/30, DlG. ll

[56] References Cited UNITED STATES PATENTS 3,198,712 8/1965 Takahashiet a1 195/30 Primary ExaminerAlvin E. Tanenholtz [57] ABSTRACT At leastone monomer selected from the group consisting of acrylamide, N,N:-loweralkylenebis(acry1amide) and his (acrylamidomethyl)ether is polymerizedin an aqueous suspension containing an aspartase-producingmicroorganism. The resultant immobilized aspartase-producingmicroorganism is subjected to enzymatic reaction with ammonium fumarateor a mixture of fumaric acid or its salt and an inorganic ammonium salt.Divalent metal ion is preferably added to the anzymatic reactionsolution. L-aspartic acid is produced.

33 Claims, No Drawings PROCESS FOR THE PRODUCTION OF L-ASPARTIC ACIDThis invention relates to a novel process for preparing L-aspartic acid.More particularly, it relates to the production of L-aspartic acid byenzymatic reaction of an immobilized aspartase-producing microorganismwith ammonium fumarate.

It is well known in the art that aspartase has the ability to convertammonium fumarate into L-aspartic acid. Various methods for producingL-aspartic acid by the enzymatic reaction of aspartase with ammoniumfumarate are known. For example, L-aspartic acid can be prepared bycultivating an aspartase-producing microorganism in a nutrient mediumcontaining fumaric acid and ammonia. Alternatively, it can be preparedby extracting aspartase from a microorganism, and reacting the enzymewith ammonium fumarate. However, these methods are disadvantageous inthe commercial production of L-aspartic acid. L-aspartic acid producedaccording to these methods is contaminated with the enzyme, mycrobialcells, nutrient sources of the medium and/or protein. Accordingly, inorder to recover L-aspartic acid having high purity additional steps forremoving the enzyme and other contaminants from the product arerequired. Furthermore, when the enzymatic reaction is completed, thereaction solution is boiled and/or acidified to produce the enzyme or anaspartase-producing microorganism, and the precipitate is filtered off.Thus, the aspartase. or the aspartase-producing microorganism can beused only once and must be discarded thereafter.

Recently, Chibata, et a1. succeeded in overcoming overcome theaforementioned disadvantages of prior art methods (Japanese PatentPublication Nos. 6870 and 17587/1970). For example, the method of Jap.Pat. Pub. No. 6870/1970 comprises binding aspartase to an anion exchangepolysaccharide adsorbent to form a substantially insoluble complex, andreacting ammonium fumarate with the aspartase complex. According to themethod of Jap. Pat. Pub. No. 17587/ 1970, L-aspartic acid can beprepared by polymerizing N,N'-methylene-bis(acrylamide) in an aqueousaspartase solution. The resultant water-insoluble aspartase preparationis then enzymatically reacted with ammonium fumarate. However, themethods of these Japanese patent publications are disadvantageous inthat they require the use of aspartase extracted from anaspartase-producing microorganism. Furthermore, denaturation of theenzyme and/or a remarkable loss in enzymatic activity inevitably occursin the course of the extraction and concentration of the extract.

As a result of various investigations, we have not found that anaspartase-producing microorganism immobilized with a semipermeablemembrane can be advantageously employed in the production of L-asparticacid.

According to the present invention, L-aspartic acid can be prepared bypolymerizing at least one monomer selected from the group consisting ofacrylamide, N,N'-lower alkylene-bis(acrylamide) and his(acrylamidomethyl)ether in an aqueous suspension contianing anaspartase-producing microorganism, and subjecting the resultantimmobilized aspartaseproducing microorganism to enzymatic reaction withammonium fumarate of a mixture of fumaric acid or its salt and aninorganic ammonium salt. The polymerization reaction of the presentinvention is preferably carried out in the presence of a polymerizationinitiator and a polymerization accelerator. Potassium persulfate,ammonium persulfate, vitamin B and Methylene Blue are suitable as thepolymerization initiator. On the other hand, B-(dimethylamino)-propionitrile and N,N,N N -tetramethylethylenediamine are employed asthe polymerization accelerator; It is preferred to carry out thereaction at 10 to 50C, especially at 20 to 40C. The reaction may becompleted within 10 to 60 minutes. Microorganisms which produceaspartase are employed for the purpose of the present invention.Examples of aspartase-producing microorganisms include Escherichia coliATCC No. 11303, Pseudomanas aeruginosa OUT (Faculty of Technology, OsakaUniversity, Japan) No. 8252, Serratia marcescens OUT No. 8259, Proteusvulgaris OUT No. 8226 [Another designation of the microorganism: FERM-P(Fermentation Research Institute, Japan) No. 526], Bacterium succinium1AM (Institute of Applied Microbiology, Tokyo University, Ja pan) No.1017 and Alcaligenesfaecalis OUT No. 8030. All of these microorganismsare publicly available from the above mentioned collections. However, itshould be noted that the present invention is not limited to the use ofthese specific microorganisms, but

includes wihtin its scope the use of all aspartaseproducingmicroorganisms. The polymerization reaction of the present inventionserves to tightly entrap each of the micro-organisms into the lattice ofthe polymer thereby affording high enzymatic activity for a long periodof time.

L-aspartic acid can be prepared by contacting the resultant immobilizedmicroorganism with ammonium fumarate or a mixture of fumaric acid or itssalt and an inorganic ammonium salt. Suitable examples of the salt offumaric acid include alkali metal salts thereof such as sodium fumarateand potassium fumarate. Ammonium chloride, ammonium sulfate and ammoniumphosphate are preferred as the inorganic ammonium salt. When a mixtureof fumaric acid or its salt and an inorganic ammonium salt is employedin the enzymatic reaction, the preferred proportion of inorganicammonium salt in the mixutre is about 1.5 to 2 moles to one mole offumaric acid or fumaric acid salt. Divalent metal ion may be added tothe enzymatic reaction solution to keep the enzymatic .activity of theimmobilized microorganism at a high level during the reaction. Forexample, when the enzymatic reaction is carried out in the presence ofdivalent metal ion, the enzymatic potency of the immobilizedmicroorganism shows no substantial depression even after one month ofsuccessive use. Suitable examples of the divalent metal ion includecalcium, magnesium, manganous and strontium ions. The preferredconcentration of divalent metal ion in the reaction solution is about0.1 to 10 milimoles/liter.

The concentration of substrate employed is not criti cal in the presentinvention. That is, ammonium fumarate or a mixture of fumaric acid orits salt and an inorganic ammonium salt is dissolved in water in anyconcentration. The solution is then adjusted to a pH of 7 to 9. Theaforementioned immobilized microorganism is suspended in the solution,and the mixture is incubated at a temperature of 25 to 45C, withstirring,

until the reaction is complete. When the reaction is completed, themixture is filtered or centrifuged to recover the immobilizedmicroorganism for subsequent use. L-aspartic acid is recovered from thefiltrate or supernatant liquid. Alternatively, the enzymatic reaction ofthe invention may be performed by a column method. The column methodenables the reaction to be carried out in a successive manner. Forexample, the immobilized microorganism is charged into a column, and anaqueous solution (pH 8 to 9) containing ammonium fumarate or a mixtureof fumaric acid or its salt and an inorganic ammonium salt is passedthrough the column at 30 to 45C, and at a suitable flow rate. An aqueoussolution containing L-aspartic acid is obtained as the effluent. Theeffluent is adjusted to pH 2.8 to 3.0 with concentrated sulfuric acid.L-aspartic acid is thereby obtained as a crystalline precipitate. Incarrying out the enzymatic reaction, the conversion rate of ammoniumfumarate to L-aspartic acid mainly depends upon the enzymatic potency ofthe immobilized microorganism, the temperature and the reaction time.With the column method, however, one can readily obtain the optimumreaction condition for complete complete conversion of ammonium fumarateto L-aspartic acid by adjusting the flow rate of the substrate solution.

In any case, during the reaction the immobilized microorganism of thepresent invention retains a high level of enzymatic activity, especiallyin the presence of divalent metal ion. Moreover, due to the sufficientdurability of the enzymic activity of the immobilized microorganism ofthe invention, repeated use of the immobilized microorganism ispossible.

Practical and presently-preferred embodiments of the present inventionwill be shown in the following Examples. In this specification, theterminology lower alkylene" should be interpreted as referring toalkylene groups having one to five carbon atoms.

EXAMPLE 1 4.8 g of the microbial cells of Escherichia coli ATCC No. l1303 are suspended in 48 ml of physiological saline solution. 9 g ofacrylamide, 480 mg of N,N'- methylene-bis (acrylamide), 6 ml of percentB-(dimethylamino)-propionitrile and 6 ml of 2.5 percent potassiumpersulfate are added to the suspension. Then, the suspension is allowedto stand at 37C for 10 minutes. 120 ml of an immobilized preparation ofEscherichia coli ATCC No. 11303 are obtained.

120 ml of the immobilized preparation of Escherichia coIiATCC No. 11303are charged into a 2.1 cm X 34.8 cm. column. 150 g of ammonium fumarateare dissolved in 900 ml of water. The aqueous solution is adjusted to pH8.5 with aqueous ammonia and diluted with water to bring the totalvolume to 1 liter. Then, the aqueous solution is continuously passedthrough the column at 37C at a flow rate of 20 ml/hr. The effluent isadjusted to pH 2.8 to 3.0 with concentrated sulfuric acid. Thecrystalline precipitate is collected by filtration and then washed withmethanol. 126.3 g of L-aspartic acid are obtained.

fab 24.8 (C =1, 6N-HCl) EXAMPLE 2 An immobilized preparation ofEscherichia coli ATCC No. 11303 is prepared in the manner described inExample 1. 50 ml of the immobilized preparation Table 1 Flow rateConversion to Laspartic acid (ml/hr) EXAMPLE 3 50 ml of an immobilizedpreparation of Escherichia coli ATCC No. 11303 prepared in the mannerdescribed in Example 1 are charged into a 2.1 cm X 14.5 cm. column. Anaqueous solution (pH 8.5) containing 1 M-concentration of ammoniumfumarate is continuously passed through the column at 37C at a flow rateas shown in Table 2. The concentration of L-aspartic acid in theeffluent obtained with the passage of time is assayed in the mannerdescribed in Example 2. The percentage conversion of ammonium fumarateto L- aspartic acid is calculated therefrom. The results are shown inTable 2.

An aqueous solution of 112.5 g of ammonium fumarate in 450 ml of wateris adjusted to pH 8.5 with aqueous ammonia and then diluted with waterto bring the total volume to 500 ml. 60 ml of an immobilized preparationof Escherichia coli ATCC No. 11303 prepared in the manner described inExample 1 are added to the aqueous solution. The mixture is stirred at37C for a period of time. The concentration of L-aspartic acid in themixture is assayed in the manner described in Example 2, and thepercentage conversion of ammonium fumarate to L-aspartic acid iscalculated therefrom. The results are shown in Table 3.

Table 3 Reaction time Conversion to L-aspartic acid After stirring for24 hours, the mixture is filtered. The filtrate is adjusted to pH 2.8 to3.0 with concentrated sulfuric acid. The crystalline precipitate iscollected by filtration and then washed with methanol. 89.7 g ofL-aspartic acid are obtained.

[ 1 24.8 (C =1, 6N-HCl) EXAMPLE 60 ml of the "mmasiiiiawrgm I)?Pseudomonas aeruginosa OUT No. 8252 are charged into a 2.1 cm X 17.4 cm.column. 500 ml of an aqueous solution (pH 8.5) containing 1M-concentration of ammonium fumarate is passed through the column at 37Cat a flow rate of ml/hr. The effluent is adjusted to pH 2.8 to 3.0 withconcentrated sulfuric acid. The crystalline precipitate is collected byfiltration and then washed with methanol. 6.31 g of L-aspartic acid areobtained.

[ 1,, 4 8 (C l, 6N-Hcn EXAMPLE 7 sulfate 7 hydrate, 4 w/v percent ofcorn steep liquor and 0.05 w/v percent of calcium carbonate. The mediumis cultivated at 37C for 20 hours under shaking. After the cultivation,the microbial cells of Escherichia coli ATCC No. l 1303 are collected bycentrifugation. The 517250551 cells aE s uspeiided H1 40 ml ofphysiological saline solution. 7.5 g acrylamide, 0.4 g of N,N'-methylene-bis(acrylamide), 5 ml of5 percentB-(dimethylamino)-propionitrile and 5 ml of 2.5 percent potassiumpersulfate are added to the suspension. Then, the suspension is allowedto stand at 37C for 10 minutes. 110 ml of an immobilized preparation ofEscherichia coli ATCC No. 11303 are obtained.

110 ml of the immobilized preparation of Escherichia coli ATCC No. 11303are charged into a 2 cm X 35 cm. column. 300 g of ammonium fumarate and0.4 g of manganous chloride 4 hydrate are dissolved in 1.8 liter ofwater. The aqueous solution is adjusted to pH 8.5 with aqueous ammoniaand diluted with water to bring the total volume to 2 liters. Then theaqueous solution is continuously passed through the column at 37C at aflow rate of 55 ml/hr. The effluent is adjusted to pH 2.8 to 3.0 withconcentrated sulfuric acid. The crystalline precipitate is collected byfiltration and then washed with cold water and methanol. 252.9 g ofL-aspartic acid are obtained.

[01],, 24.8 (C =1, 6N-HCl) EXAMPLE 8 ml of an immobilized preparation ofEscherichia coli ATCC No. 11303 prepared in the manner de- Table 4Operation tim e Conversion of ammonium fumarate to L-aspartic acid Metalion added No addition 2.4 g of the microbial cells of Serratiamarcescens OUT No. 8259 are treated in the manner described in ExampleS.60 ml of an immobilized preparation of Serratia marcescens OUT No. 8259are obtained. A mixture of 60 ml of the immobilized preparation, 58 g offumaric acid and 37.1 g of ammonium chloride is added to 450 ml ofwater. The mixture is adjusted to pH 8.5 with an aqueous l N-sodiumhydroxide solution and then diluted with water to bring the total volumeto 500 ml. The mixture is stirred at 37C for 24 hours The mixture isfiltered. The filtrate is adjusted to pH 2.8 to 3.0 with concentratedsulfuric acid. The crystalline precipitate is collected by filtrationand washed with methanol. 62.9 g of L-aspartic acid are obtained.

EXAMPLE 9 50 ml of an immobilized preparation of Escherichia coli ATTCNo. 11303 prepared in the manner described in Example 7 are charged intoa 2 cm X 16 cm.

column. An aqueous solution (pH 8.5) containing 1 M-concentration ofammonium fumarate and 1 mM- concentration of manganous ion iscontinuously passed through the column at 37C at a flow rate as shown inTable 5. The concentration of L-aspartic acid in the effluent is assayedin the manner described in Example 2, and the percentage conversion ofammonium fumarate to L-aspartic acid is calculated therefrom. Theresults are shown in Table 5.

An aqueous solution of 150 g of ammonium fumarate and 0.2 g of manganouschloride 4 hydrate in 900 ml of water is adjusted to pH 8.5 with aqueousammonia and then diluted with water to bring the total volume to oneliter. 100 ml of an immobilized preparation of Escherichia coli ATCC No.11303 prepared in the manner described in Example 7 are added to theaqueous solution. The mixture is stirred at 37C for a period of time.The concentration of L-aspartic acid in the mixture is assayed in themanner described in Example 2, and the percentage conversion of ammoniumfumarate to L-aspartic acid is calculated therefrom. The results areshown in Table 6.

After stirring for 10 hours, the mixture is filtered. The filtrate isadjusted to pH 2.8 to 3.0 with concentrated sulfuric acid. Thecrystalline precipitate is collected by filtration and then washed withcold water and methanol. 126.4 g of L-aspartic acid are obtained.

[ 24.8 (c 1, 6N-HCl) EXAMPLE n Pseudomonas aeruginosa OUT No. 8252 isinoculated into one liter of a medium (pH 7.0) containing 3 w/v percentof sodium fumarate, 2 w/v percent of ammonium sulfate, 0.2 w/v percentof peptone and 0.5 w/v percent of yeast extract. The medium iscultivated at 37C for 20 hours under shaking. After the cultivation,

the microbial cells of Pseudomonas aeruginosa OUT No. 8252 are collectedby centrifugation. The microbial cells are suspended in 48 ml ofphysiological saline solution. 9 g of acrylamide, 0.48 g ofN,N-methylenebis(acrylamide), 6 ml of percent B-(dimethylamino)-propionitrile and 6 ml of 2.5 percent potassium persulfate are added tothe suspension. Then, the suspension is allowed to stand at 37C forminutes. 120 ml of an immobilized preparation of Pseudomonas aeruginosaOUT No. 8252 are obtained.

ml of the immobilized preparation of Pseudomonas aeruginosa OUT No. 8252are charged into a 2 cm X 38 cm column. One liter of an aqueous solution(pH 8.5) containing 1 M-concentration of ammonium fumarate and lmM-concentration of manganous ion is continuously passed through thecolumn at 37C at a flow rate of 20 ml/hr. The effluent is adjusted to pH2.8 to 3.0 with concentrated sulfuric acid. The crystalline precipitateis collected by filtration and then washed with cold water and methanol.l26.0 g of L-aspartic acid are obtained.

[ 24.8 c 1, 6NHCl) EXAMPLE 12 Serratia marcescens OUT No. 8259 isinoculated into one liter of a medium (pH 7.0) containing 3 w/v percentof sodium fumarate, 2 w/v percent of ammonium sulfate, 0.2 w/v percentof peptone and 0.5 w/v percent of yeast extract. The medium iscultivated at 37C for 20 hours under shaking. After the cultivation, themicrobial cells of Serratia marcescens OUT No. 8259 are collected bycentrifugation. The microbial cells are suspended in 32 ml ofphysiological saline solution. 6 g of acrylamide, 0 .32 g ofN,N-methylenebis(acrylamide), 4 ml of5 percent B-(dimethylamino)-propionitrile and 4 ml of 2.5 percent potassium persulfate are added tothe suspension. Then, the suspension is allowed to stand at 37C for 10minutes. 100 ml of an immobilized preparation of Serratia marcescens OUTNo. 8259 are obtained.

100 ml of the immobilized preparation of Serratia macrescens OUT No.8259 are added to one liter of an aqueous solution (pH 8.5) containing gof sodium fumarate, 4.2 g of ammonium chloride and 0.2 g of manganouschloride 4 hydrate. The mixture is stirred at 37C for 24 hours. Themixture is filtered. The filtrate is adjusted to pH 2.8 to 3.0 withconcentrated sulfuric acid. The crystalline precipitate is collected byfiltration and then washed with cold water and methanol. 126.4 g ofL-aspartic acid are obtained.

[11],, 24.8 (C l, 6N-HCl) EXAMPLE 13 2.4 g of the microbial cells ofEscherichia coli ATCC No. l 1303 are suspended in 24 ml of physiologicalsaline solution. 4.5 g of acrylamide, 240 mg of N,N'-methylene-bis(acrylamide), 3 ml of5 percentB-(dimethylamino)-propionitrile and 3 ml of 2.5 percent potassiumpersulfate are added to the suspension. Then, the suspension is allowedto stand at 37C for 30 minutes. 60 ml of an immobilized preparation ofEscherichia coli ATCC No. 11303 are obtained.

60 ml of the immobilized preparation of Escherichia coli ATCC No. 1 1303are added to 500 ml of an aqueous solution (pH 8.5) containing 1M-concentration of ammonium fumarate and l mM-concentration of manganousion. The mixture is stirred at 37C for a period of time. Theconcentration of L-aspartic acid in the mixture is assayed in the mannerdescribed in Example 2, and the percentage conversion of ammoniumfumarate to L-aspartic acid is calculated therefrom. The

results are shown in Table 7.

Table 7 Table 9 Reaction time Conversion to L-aspartic acid Reactiontime Conversion to L-aspartic acid (hr.) (hr.)

57 5 5 3o 8 80 8 61 I0 100 10 a2 100 EXAMPLE 14 f l h CC EXAMPLE 16 2.4g. o the microbia cells of Esc erichia coli AT 7 No. 11303 are suspendedin 24 ml of a physiological g of the microbial cell? of Eschenchla i TNo. 11303 are suspended 1n 24 ml of a physiological saline solution. 4.5g of acrylamide, 240 mg of N,N

l5 saline solution. 60 mg of N,N -metl1ylenepropylene-bis(acrylamide), 3ml of 5 percent B-(dimethylamino)-propionitrile and 3 ml of 2.5 percentpotassium persulfate are added to the suspension. Then, the suspensionis allowed to stand at 37C for 30 minutes. 57 ml of an immobilizedpreparation of Escherichia coli ATCC No. 11303 are obtained.

57 ml of the immobilized preparation of Escherichia coli ATCC No. 11303are added to 500 ml of an aqueous solution (pH 8.5) containing 1M-concentration of ammonium fumarate and 1 mM-concentration of manganousion. The mixture is stirred at 37C for a pe riod of time. Theconcentration of L-aspartic acid in the mixture is assayed in the mannerdescribed in Example 2, and the percentage conversion of ammoniumfumarate to L-aspartic acid is calculated therefrom. The results areshown in Table 8.

minutes.

bis(acrylamide), 1.8 ml of 0.112 percent N,N,N',N-tetramethylethylenediamine and 0.2 ml of 2.5 percent ammonium persulfateare added to the suspension. Then, the suspension is allowed to stand at37C for 60 36 ml of an immobilized preparation of Escherichia coli ATCCNo. 11303 are obtained.

36 ml of the immobilized preparation of Escherichia coli ATCC No. 11303are added to 500 ml of an aqueous solution (pH 8.5) containing 1M-concentration of ammonium fumarate and l mM-concentration of manganousion. The mixture is stirred at 37C for a period of time. Theconcentration of L-aspartic acid in the mixture is assayed in the mannerdescribed in Example 2, and the percentage conversion of ammoniumfumarate to L-aspartic acid is calculated therefrom. The results areshown in Table 10.

Table 8 Table 10 Reaction time Conversion to L-aspartic acid Reactiontime Conversion to L-aspartic acid (hr.) (hr.)

EXAMPLE 15 EXAMPLE l7 g of tha microbial cells of Escherichia ATCC 2.4oi t li microbial cells of Escherichia coli ATCC 11303 are Suspended in24 ml of a physiological No. 11303 are suspended in 24 ml ofphysiological sasaline solution. 4.5 g of acrylamide, 240 mg of bis(alil i 60 mg f N N' 1 y t y ether. 3 ml of 5 p cent B-( bis(acrylamide),1.8 ml of 0.112 percent N,N,N',N'- thylamino)-propionitrile and 3 ml of2.5 percent potastetramethylenediamine and 0.2 ml of 2.5 percent amsiumpersulfate are added to the suspension. Then, the monium persulfate areadded to the suspension. Then, suspension is allowed to stand at 37C for30 minutes. the suspension is allowed to stand at 37C for min- 52 ml ofthe immobilized preparation of Escherichia utes. 42 ml of an immobilizedpreparation of coli ATCC No. 11303 are obtained. Escherichia coli ATCCNo. 11303 are obtained.

52 ml of the immobilized preparation of Escherichia 42 ml of theimmobilized preparation of Escherichia coli ATCC No. 11303 are added to500 ml of an aquecoli ATCC No. ll303 are added to 500 ml of an aqueoussolution (pH 8.5) containing 1 M-concentration of 60 0B8 Solution (PContaining l M-co centration of ammonium fumarate and l mM-concentrationof manammonium fumarate and l IM-Co entration 0f ganous ion. The mixtureis stirred at 37C for a period manganous The mixture is t red at 37C fora pef time The concentration f L aspartic id i h riod of time. Theconcentration of L-aspartic acid in mixture is assayed in the mannerdescribed in Example the mixture is assayed in the manner descfibed in2, and the percentage conversionofammoniuinEmarample and the PercentageConverslo" of ammmllum ate to L-aspartic acid is calculated therefrom.The results are shown in Table 9.

fumarate to L-aspartic acid is calculated therefrom. The results areshown in Table 11.

Ta ble l l V 7 Reaction time Conversion to L-aspartic acid EXAMPLE 182.4 g of the microbial cells of Escherichia coli ATCC No. 11303 aresuspended in 24 ml of a physiological saline solution. 60 mg ofbis(acrylamidomethyl)ether, 1.8 ml of 0.1 12 percentN,N,N',N'-tetramethylethylenediamine and 0.2 ml of 2.5 percent ammoniumpersulfate are added to the suspension. Then, the suspension is allowedto stand at 37C for 60 minutes. ml of an immobilized preparation ofEscherichia coli ATCC No. 11303 are obtained.

35 ml of the immobilized preparation of Escherichia coli ATCC No. 11303are added to 500 ml of an aqueous solution (pH 8.5) containing 1M-concentration of ammonium fumarate and 1 mM-concentration of manganousion. The mixture is stirred at 37C for a period of time. Theconcentration of L-aspartic acid in the mixture is assayed in the mannerdescribed in Example- 2, and the percentage conversion of ammoniumfumarate to L-aspartic acid is calculated therefrom. The results areshown in Table 12.

Table 12 Reaction time Conversion to L-aspartic acid hat w laims 3. Theprocess according to claim 1, wherein acrylamide andbis(acrylamidomethyl)ether are polymerized.

4. The process according to claim 1, wherein N,N'- loweralkylene-bis(acrylamide) or bis(acrylamidomethyl)ether are polymerized.

5. The process according to claim 1, wherein the polymerization iscarried out in the presence of a polymerization initiator and apolymerization accelerator at 10 to 50C.

6. The process according to claim 1, wherein the enzymatic reaction iscarried out at 25 to 45C, at pH 7 to 9.

7. A process for preparing L-aspartic acid which comprisescopolymerizing acrylamide and N,N'-lower alkylene-bis(acrylamide) in anaqueous suspension of an aspartase-producing microorganism at 10 to 50Cin the presence of a polymerization initiator and a polymerizationaccelerator to produce an immobilized aspartase-producing microorganism,and subjecting the immobilized aspartase-producing microorganism toenzymatic reaction with ammonium fumarate or a mixture of fumaric acidor its salt and an inorganic ammonium salt at 25 to 45C, at pH 7 to 9.

8. A process for preparing L-aspartic acid which comprisescopolymerizing acrylamide and bis(acrylamidomethyl)ether in an aqueoussuspension of an aspartase-producing microorganism at 10 to 50C in thepresence of a polymerization initiator and a polymerization acceleratorto produce an immobilized aspartase-producing microorganism, andsubjecting the immobilized aspartase-producing microorganism toenzymatic reaction with ammonium fumarate or a mixture of fumaric acidor its salt and an inorganic ammonium salt at 25 to 45C, at pH 7 to 9.

9. A process for preparing L-aspartic acid which comprises polymerizingN,N-lower alkylenebis(acrylamide) or bis (acrylamidomethyl)ether in anaqueous suspension of an aspartase-producing microorganism at 10 to 50Cin the presence of a polymerization initiator and a polymerizationaccelerator to produce an immobilized aspartase-producing microorganism,and subjecting the immobilized aspartaseproducing microorganism toenzymatic reaction with ammonium fumarate or a mixture of fumaric acidor its salt and an inorganic ammonium salt at 25 to 45C, at pH 7 to 9.

10. The process according to claim 5, wherein the polymerizationinitiator is selected from the group consisting of potassium persulfate,ammonium persulfate, vitamin B and methylene blue.

11. The process according to claim 7, wherein the polymerizationinitiator is selected from the group consisting of potassium persulfate,ammonium persulfate, vitamin B and methylene blue.

12. The process according to claim 8, wherein the polymerizationinitiator is selected from the group consisting of potassium persulfate,ammonium persulfate, vitamin B and methylene blue.

13. The process according to claim 9, wherein the polymerizationinitiator is selected from the group consisting of potassium persulfate,ammonium persulfate, vitamin B and methylene blue.

14. The process according to claim 5, wherein the polymerizationaccelerator is selected from the group consisting ofB-(dimethylamino)-propionitrile and N,-N,N',N-tetra-methylethylenediamine.

15. The process according to claim 7, wherein the polymerizationaccelerator is selected from the group consisting ofB-(dimethylamino)-propionitrile and N,-N,N,N'-tetra-methylethylenediamine.

16. The process according to claim 8, wherein thepolymerizationaccelerator is selected from the group 13 consisting ofB-(dimethylamino)-propionitrile and N,-N,N',N'-tetra-methylethylenediamine.

17. The process according to claim 9, wherein the polymerizationaccelerator is selected from the group consisting ofB-(dimethylamino)-propionitrile and N,-

N,N,N-tetra-methylethylenediamine.

18. The process according to claim 1, wherein the salt of fumaric acidis selected from the group consisting of sodium fumarate and potassiumfumarate.

19. The process according to claim 7, wherein the salt of fumaric acidis selected from the group consisting of sodium fumarate and potassiumfumarate.

20. The process according to claim 8, wherein the salt of fumaric acidis selected from the group consisting of sodium fumarate and potassiumfumarate'.

21. The process according to claim 9, wherein the salt of fumaric acidis selected from the group consisting of sodium fumarate and potassiumfumar'ate.

22. The process according to claim 1, wherein the inorganic ammoniumsalt is selected from the group consisting of ammonium chloride,ammonium sulfate and ammonium phosphate.

23. The process according to claim 7, wherein the inorganic ammoniumsalt is selected from the group consisting of ammonium chloride,ammonium sulfate and ammonium phosphate.

24. The process according to claim 8, wherein the inorganic ammoniumsalt is selected from the group consisting of ammonium chloride,ammonium sulfate and ammonium phosphate.

25. The process according to claim 9, wherein the inorganic ammoniumsalt is selected from the group consisting of ammonium chloride,ammonium sulfate and ammonium phosphate.

26. The process according to claim 1, wherein the enzymatic reaction iscarried out in the presence of 0.1 to 10 millimoles/liter of a divalentmetal ion.

27. The process according to claim 7, wherein the enzymatic reaction iscarried out in the presence of 0.1 to 10 millimoles/liter of a divalentmetal ion.

28. The process according to claim 8, wherein the enzymatic reaction iscarried out in the presence of 0.1 to 10 millimoles/liter of a divalentmetal ion.

29. The process according to claim 9, wherein the enzymatic reaction iscarried out in the presence of 0.1 to 10 millimoles/liter of a divalentmetal ion.

30. The process according to claim 26, wherein the divalent metal ion isselected from the group consisting of calcium, magnesium, manganous andstrontium ion.

31. The process according to claim 27, wherein the divalent metal ion isselected from the group consisting of calcium, magnesium, manganous andstrontium ion.

33. The process according to claim 29, wherein the divalent metal ion isselected from the group consisting of calcium, magnesium, manganous andstrontium ion.

2. The process according to claim 1, wherein acrylamide and N, N''-loweralkylene-bis(acrylamide) are polymerized.
 3. The process according toclaim 1, wherein acrylamide and bis(acrylamidomethyl)ether arepolymerized.
 4. The process according to claim 1, wherein N,N''-loweralkylene-bis(acrylamide) or bis(acrylamidomethyl)ether are polymerized.5. The process according to claim 1, wherein the polymerization iscarried out in the presence of a polymerization initiator and apolymerization accelerator at 10* to 50*C.
 6. The process according toclaim 1, wherein the enzymatic reaction is carried out at 25* to 45*C,at pH 7 to
 9. 7. A process for preparing L-aspartic acid which comprisescopolymerizing acrylamide and N,N''-lower alkylene-bis(acrylamide) in anaqueous suspension of an aspartase-producing microorganism at 10* to50*C in the presence of a polymerization initiator and a polymerizationaccelerator to produce an immobilized aspartase-producing microorganism,and subjecting the immobilized aspartase-producing microorganism toenzymatic reaction with ammonium fumarate or a mixtUre of fumaric acidor its salt and an inorganic ammonium salt at 25* to 45*C, at pH 7 to 9.8. A process for preparing L-aspartic acid which comprisescopolymerizing acrylamide and bis(acrylamidomethyl)ether in an aqueoussuspension of an aspartase-producing microorganism at 10* to 50*C in thepresence of a polymerization initiator and a polymerization acceleratorto produce an immobilized aspartase-producing microorganism, andsubjecting the immobilized aspartase-producing microorganism toenzymatic reaction with ammonium fumarate or a mixture of fumaric acidor its salt and an inorganic ammonium salt at 25* to 45*C, at pH 7 to 9.9. A process for preparing L-aspartic acid which comprises polymerizingN,N''-lower alkylene-bis(acrylamide) or bis (acrylamidomethyl)ether inan aqueous suspension of an aspartase-producing microorganism at 10* to50*C in the presence of a polymerization initiator and a polymerizationaccelerator to produce an immobilized aspartase-producing microorganism,and subjecting the immobilized aspartase-producing microorganism toenzymatic reaction with ammonium fumarate or a mixture of fumaric acidor its salt and an inorganic ammonium salt at 25* to 45*C, at pH 7 to 9.10. The process according to claim 5, wherein the polymerizationinitiator is selected from the group consisting of potassium persulfate,ammonium persulfate, vitamin B2 and methylene blue.
 11. The processaccording to claim 7, wherein the polymerization initiator is selectedfrom the group consisting of potassium persulfate, ammonium persulfate,vitamin B2 and methylene blue.
 12. The process according to claim 8,wherein the polymerization initiator is selected from the groupconsisting of potassium persulfate, ammonium persulfate, vitamin B2 andmethylene blue.
 13. The process according to claim 9, wherein thepolymerization initiator is selected from the group consisting ofpotassium persulfate, ammonium persulfate, vitamin B2 and methyleneblue.
 14. The process according to claim 5, wherein the polymerizationaccelerator is selected from the group consisting of Beta-(dimethylamino)-propionitrile andN,N,N'',N''-tetra-methylethylenediamine.
 15. The process according toclaim 7, wherein the polymerization accelerator is selected from thegroup consisting of Beta -(dimethylamino)-propionitrile andN,N,N'',N''-tetra-methylethylenediamine.
 16. The process according toclaim 8, wherein the polymerization accelerator is selected from thegroup consisting of Beta -(dimethylamino)-propionitrile andN,N,N'',N''-tetra-methylethylenediamine.
 17. The process according toclaim 9, wherein the polymerization accelerator is selected from thegroup consisting of Beta -(dimethylamino)-propionitrile andN,N,N'',N''-tetra-methylethylenediamine.
 18. The process according toclaim 1, wherein the salt of fumaric acid is selected from the groupconsisting of sodium fumarate and potassium fumarate.
 19. The processaccording to claim 7, wherein the salt of fumaric acid is selected fromthe group consisting of sodium fumarate and potassium fumarate.
 20. Theprocess according to claim 8, wherein the salt of fumaric acid isselected from the group consisting of sodium fumarate and potassiumfumarate.
 21. The process according to claim 9, wherein the salt offumaric acid is selected from the group consisting of sodium fumarateand potassium fumarate.
 22. The process according to claim 1, whereinthe inorganic ammonium salt is selected from the group consisting ofammonium chloride, ammonium sulfate and ammonium phosphate.
 23. Theprocess according to claim 7, wherein the inorganic ammonium salt isselected from the group consisting of ammonium chloride, ammoniumsulfate and ammonium phosphate.
 24. The process according to claim 8,wherein the inorganic ammOnium salt is selected from the groupconsisting of ammonium chloride, ammonium sulfate and ammoniumphosphate.
 25. The process according to claim 9, wherein the inorganicammonium salt is selected from the group consisting of ammoniumchloride, ammonium sulfate and ammonium phosphate.
 26. The processaccording to claim 1, wherein the enzymatic reaction is carried out inthe presence of 0.1 to 10 millimoles/liter of a divalent metal ion. 27.The process according to claim 7, wherein the enzymatic reaction iscarried out in the presence of 0.1 to 10 millimoles/liter of a divalentmetal ion.
 28. The process according to claim 8, wherein the enzymaticreaction is carried out in the presence of 0.1 to 10 millimoles/liter ofa divalent metal ion.
 29. The process according to claim 9, wherein theenzymatic reaction is carried out in the presence of 0.1 to 10millimoles/liter of a divalent metal ion.
 30. The process according toclaim 26, wherein the divalent metal ion is selected from the groupconsisting of calcium, magnesium, manganous and strontium ion.
 31. Theprocess according to claim 27, wherein the divalent metal ion isselected from the group consisting of calcium, magnesium, manganous andstrontium ion.
 32. The process according to claim 28, wherein thedivalent metal ion is selected from the group consisting of calcium,magnesium, manganous and strontium ion.
 33. The process according toclaim 29, wherein the divalent metal ion is selected from the groupconsisting of calcium, magnesium, manganous and strontium ion.